Tài liệu Báo cáo Y học: NMR-based determination of the binding epitope and conformational analysis

NMR-based determination of the binding epitope

and conformational analysis of MUC-1 glycopeptides and peptides

bound to the breast cancer-selective monoclonal antibody SM3

Heiko Mo¨ ller1

, Nida Serttas1

, Hans Paulsen1

, Joy M. Burchell2

, Joyce Taylor-Papadimitriou2

and Bernd Meyer1

1

Institute of Organic Chemistry, University of Hamburg, Germany; 2

Imperial Cancer Research Fund Breast Cancer Biology Group,

Guy’s Hospital, London, UK

Mucin glycoproteins on breast cancer cells carry shortened

carbohydrate chains. These partially deglycosylated mucin 1

(MUC-1) structures are recognized by the monoclonal

antibody SM3, which is being tested for its diagnostic utility.

We used NMR spectroscopy to analyze the binding mode

and the binding epitope of peptide and glycopeptide antigens

to the SM3 antibody. The pentapeptide PDTRP and the

glycopentapeptide PDT(O-a-D-GalNAc)RP are known lig￾ands of the monoclonal antibody. The 3D structures of the

ligands in the bound conformation were determined by an￾alyzing trNOESY build-up rates. The peptide was found to

adopt an extended conformation that fits into the binding

pocket of the antibody. The binding epitopes of the ligands

were determined by saturation transfer difference (STD)

NMR spectroscopy. The peptide’s epitope is predominantly

located in the N-terminal PDT segment whereas the C-ter￾minal RP segment has fewer interactions with the protein.

In contrast, the glycopeptide is interacting with SM3

utilizing all its amino acids. Pro1 shows the strongest binding

effect that slightly decays towards Pro5. The GalNAc resi￾due interacts mainly via the N-acetyl residue while the other

protons show less interactions similar to that of Pro5. The

glycopeptide in the bound state also has an extended con￾formation of the peptide with the carbohydrate oriented

towards the N-terminus. Docking studies showed that pep￾tide and glycopeptide fit the binding pocket of the mAb SM3

very well.

Keywords: glycopeptide antibody complex; STD NMR;

breast cancer; MUC-1; binding epitope.

The extracellular part of the epithelial glycoprotein MUC-1

consists of tandem repeats of 20 amino acids

(PDTRPAPGSTAPPAHGVTSA, where the start of the

tandem repeat peptide sequence varies. We follow here the

definition by Gendler et al. who defined the start at PDTRP

[1]. Residues of peptides, that were elongated at the

N-terminus, are designated by an apostrophe, e.g. Ala20¢-

Pro1-Asp2-Thr3-Arg4-Pro5.) [2]. Each repeat can carry up

to five O-glycosyl chains at Ser and Thr residues that

account for the high carbohydrate content of the mucins [3].

Usually, 70–100 repeats are found in mucins. The clustering

of O-linked glycans on MUC-1 leads to an extended protein

core. Membrane-bound mucins extend several hundred

nanometers into the lumen and thus represent a first barrier

to the environment. They have important functions in cell￾cell recognition and shield the cell from microorganisms,

toxins and proteolytic attack [4].

Many diseases affect the production of mucus. Both the

amount and the characteristics of the mucus can be altered.

In cystic fibrosis, for example, due to changes of the ionic

environment dramatic alterations in rheological properties

correlate with changes in carbohydrate composition [2].

Modified oligosaccharides are also found in mucins of

patients with Crohn’s disease [2].

Epithelial cells express the membrane-bound MUC-1 at

their apical surface. In carcinomas, the localization at the

apical surface is lost. High concentrations of MUC-1 spread

out over the whole cell surface. This may protect the cells

against low pH and may interfere with immune surveillance

by causing steric hindrance of surface antigen presentation

[2,4].

In breast cancer, the MUC-1 glycoprotein is overex￾pressed and aberrantly glycosylated. Thus, in contrast to the

mucin produced by normal breast epithelial cells, which

carry core2 based structures [5], MUC1 from breast cancer

cells carries highly truncated, mainly core 1 based oligosac￾charide structures [6,7]. In some cases, the first sugar added,

N-acetyl galactosamine is not extended, or is sialylated to

form the cancer-related sialyl Tn epitope. Because of the

shorter side chains, the peptide core of the cancer mucin is

more exposed, and antibodies have been developed which

recognize epitopes exposed in the cancer mucin, which are

normally masked by large oligosaccharide side chains.

These antigenic peptide sequences therefore constitute

cancer-associated epitopes which are also found in the

Correspondence to B. Meyer, Institute of Organic Chemistry,

University of Hamburg, Martin-Luther-King-Platz 6,

20146 Hamburg, Germany.

Fax: + 49 (0)40 42838 2878, Tel.: + 49 (0)40 42838 5913,

E-mail: [email protected]

Abbreviations: MUC-1, mucin 1 glycoprotein; SM3, breast cancer￾selective monoclonal antibody; STD NMR, saturation transfer

difference NMR; trNOE, transferred nuclear Overhauser enhance￾ment; SPR, surface plasmon resonance; SAR, structure activity

relationship; MD, molecular dynamics.

(Received 28 August 2001, revised 5 December 2001, accepted

14 January 2002)

Eur. J. Biochem. 269, 1444–1455 (2002) Ó FEBS 2002